1. Field of the Invention
The present invention relates to a isopentenyl diphosphate isomerase from Hevea Brasiliensis and a rubber producing method using the same.
2. Description of the Prior Art
The isoprenoid biosynthetic pathway is ubiquitous to all living organisms and produces more than 23,000 compounds which play vital roles in the structure of cells, electron transport, photosynthesis, cell-to-cell signaling, and interactions between organisms (Ramos-Valdivia et al., 1997). Several important classes of compounds derived from this complex pathway include sterols, carotenoids, dolichols, ubiquinones, and prenylated proteins (Hahn and Poulter, 1995). All of these compounds are derived from the same building block, IPP. In most eukaryotes, IPP is synthesized from three molecules of acetyl-CoA via the mevalonic acid pathway(Chappell, 1995). Recently, a separate mevalonate-independent pathway was discovered in bacteria and plant(Lichtenthaler et al., 1997; Romer et al., 1993). This pathway utilizes three-carbon precursors such as glyceraldehyde phosphate, pyruvate, and dihydroxyacetone phosphate. The enzyme IPP isomerase (hereafter "IPI") catalyzes the interconversion of IPP to its highly electrophilic isomer, dimethylallyl diphosphate (DMAPP). These two isomers serve as substrates for the synthesis of isoprenoid compounds.
Bioengineering of isoprenoid biosynthesis is of keen interest because of the commercial value of plant compounds such as essential oils, pharmaceuticals, rubber, and waxes. Although the interconversion of IPP and DMAPP is catalyzed by the same IPP isomerase, the equilibrium is in favor of DMAPP production(Sun et al., 1998). This isomerization reaction may be a rate-limiting step for isoprenoid biosynthesis. Expression of an exogenous IPI gene enhanced isoprenoid biosynthesis in E. coli (Kajiwara et al., 1997). Transgenic tobacco plants transformed with a H. brasiliensis HMGCoA reductase over-produced total sterols up to six-times than the control plants did (Schaller et al., 1995).
Natural rubber (cis-1,4-polyisoprene) is an important raw material for many industrial uses. Although rubber is produced in about 2,000 plant species(Backhaus, 1985), H. brasiliensis has been the only commercial source of natural rubber mainly due to its abundance in the tree, its quality and the ease of harvesting. Diminishing acreage of rubber plantation coupled with increasing demand has renewed research interests on the study of rubber biosynthesis and the development of an alternative rubber source. The first step in rubber biosynthesis is the isomerization of IPP to DMAPP by IPP isomerase. The successive head-to-tail condensation reactions of the five-carbon intermediates catalyzed by enzyme(s) referred to as rubber transferase (or polymerase) have been assumed to yield rubber. IPP isomerase activity has been found in bottom fraction and C-serum of rubber latex of H. brasiliensis (Koyama et al., 1996; Tangpakdee et al., 1997). The presence of a site-directed specific inhibitor of IPP isomerase, 3,4-oxido-3-methyl-1-butyl diphosphate (OMBPP), inhibited incorporation of IPP into rubber in in vitro rubber assay (Cornish, 1993).
IPP isomerase catalyzes the conversion of IPP to DMAPP, which is an essential step in the biosynthesis of all isoprenoids including carotenoids, growth regulators, and natural rubber. The enzyme from a wide variety of organisms has been studied for a review, see (Ramos-Valdivia et al., 1997). In higher plants, IPP isomerase has been purified and characterized from Capsicum chromoplasts(Dogbo and Camara, 1987), Cinchona robusta cell suspension (Ramos-Valdivia et al., 1997), H. brasiliensis (Koyama et al., 1996). To date relatively few IPP isomerase sequences from higher plants appear in the literature. Full-length genes for higher plant IPP isomerase are known only for Arabidopsis(Campbell et al., 1997) and two flowering plants Clarkia spp.(Blanc et al., 1996; Blanc and Pichersky, 1995). Partial sequence is also available for tobacco (Accession No. Y09634). However, none of the genes has been implicated in rubber biosynthesis.
As the isomerization of IPP to DMAPP is the first step in rubber biosynthesis, it is plausible for IPP isomerase to play a key role in rubber biosynthesis. Although IPP isomerase activity was observed in both bottom and C-serum fractions from Hevea latex (Tangpakdee et al., 1997), neither the protein nor cDNA encoding IPP isomerase has been isolated and studied from Hevea rubber tree.